The CD4 coreceptor molecule is essential for the generation and function of T helper-lineage cells. Its expression is strictly regulated during T cell development. Transcription of the CD4 gene is repressed in T progenitor cells, then upregulated in immature thymocytes, and persists in T helper cells, but importantly, it is silenced in thymocytes committed to the cytotoxic lineage. Studies in gene-targeted mice have demonstrated that CD4 silencing is established through a stepwise process, beginning from CD4-silencer dependent gene repression to silencer-independent epigenetic silencing. Our recent data suggest that establishment of CD4 silencing is regulated by cooperative interactions of components of the CD4 silencing complexes with the CD4 silencer. The long-term goal of the proposed studies is to understand the molecular mechanisms that convey the lineage specificity of CD4 silencing and its epigenetic inheritance. Findings from the proposed studies are likely to advance our understanding of cell fate determination. Specific Aim 1 seeks to biochemically characterize the protein components responsible for CD4 silencing. Using both DNA affinity chromatography and immuno-affinity purification methods, candidate protein subunits will be isolated for peptide sequencing. In addition, the potential role of differential posttranslational modifications of Runx3 and its co-factor CBFbeta in different T-lineage cells will be examined. Specific Aim 2 will investigate the role of heterochromatin packaging of the CD4 locus in permanent CD4 silencing. Since the CD4 locus is associated with centromeric heterochromatin clusters in CD8+ but not in CD4+ cells, it is conceivable that heterochromatin packaging epigenetically maintains CD4 silencing. This hypothesis will be tested in a mouse model in which the CD4 locus can be directed to or excluded from the centromeric heterochromatin structures. Specific Aim 3 will test the hypothesis that nuclear translocation of CBFbeta is required for initiating lineage specific CD4 silencing. The transcription factor Runx3 has been shown to be one of the key players in the initiation of CD4 silencing. Runx proteins bind to DNA as a heterodimer with CBFbeta. We have found that CBFbeta exhibits T-lineage specific subcellular localization. The distinct pattern of CBFbeta subcellular localization also correlates well with the affinity of Runx proteins for DNA and silencing activity. Here, we will determine whether the level of nuclear CBFbeta proteins in CD4+ and CD8+ T cells may affect CD4 silencing, by overexpression or RNAi blocking experiments.